Localization of an insulin-like growth factor (IGF) binding site of bovine IGF binding protein-2 using disulfide mapping and deletion mutation analysis of the C-terminal domain.
We have investigated which region(s) of bovine insulin-like growth factor binding protein-2 (bIGFBP-2) interact with insulin-like growth factors (IGFs) using C-terminally truncated forms of bIGFBP-2. Initially to aid in mutant design, we defined the disulfide bonding pattern of bIGFBP-2 C-terminal region using enzymatic digestion. The pattern is Cys186-Cys220, Cys231-Cys242, and Cys244-Cys265. In addition, cyanogen bromide cleavage of bIGFBP-2 revealed that the N- and C-terminal cysteine-rich domains were not linked by disulfide bonds. Taking the disulfide bonding pattern into consideration, C-terminal truncation mutants were designed and expressed in COS-1 mammalian cells. Following IGF binding assays, a region between residues 222 and 236 was identified as important in IGF binding. Specifically, mutants truncated by 14, 36, and 48 residues from the C terminus bound IGFs to the same extent as wild type (WT) bIGFBP-2. Removal of 63 residues resulted in a greatly reduced (up to 80-fold) ability to bind IGF compared with WT bIGFBP-2. Interestingly this mutant lacked the IGF-II binding preference of WT bIGFBP-2. Residues 236-270 also appeared to play a role in determining IGF binding specificity as their removal resulted in mutants with higher IGF-II binding affinity.
Forbes
B E
BE
Cooperative Research Centre for Tissue Growth and Repair, P. O. Box 10065, Gouger St., Adelaide, South Australia 5005, Australia.
Turner
D
D
Hodge
S J
SJ
McNeil
K A
KA
Forsberg
G
G
Wallace
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry*; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010449:Peptide Mapping; D017384:Sequence Deletion; D013002:Somatomedins / Q000378:metabolism*; D014357:Trypsin / Q000737:chemistry
D004220:Disulfides; D018971:Insulin-Like Growth Factor Binding Protein 2; D013002:Somatomedins; D014357:Trypsin